The genetic analysis of tumorigenesis is being carried out with the uniquely useful stably diploid, nontumorigenic CHEF/18 cell line (Chinese hamster embryo fibroblast), which was developed in this laboratory. We previously have shown that CHEF/18 cells do not form tumors after single- step mutagenesis, requiring multiple genetic changes to become tumorigenic. We have shown further that CHEF cells are excellent recipients of transfected plasmid or genomic DNAs derived from tumors and give rise to foci containing tumorigenic cells. This system is being used to determine how many different genes are involved in the tumorigenic transformation of this single cell type, which forms spindle-cell sarcomas in nude mice. For this purpose, we have developed a library of transformed and tumor-derived lines from CHEF/18 using a variety of carcinogenic procedures including chemical carcinogenesis, exposure to 5-azacytidine, transfection with cloned oncogenes, and most recently, "hit and run" tumorigenesis with particular vectors: pSVgpt and J132 (containing the Moloney LTR and the normal human H-ras gene). Several of these cell lines have provided genomic DNA that induces foci in CHEF/18 recipients, and they are now being screened by genomic cloning and sib selection to identify the transforming sequences. Transformed foci of CHEF/18 cells undergo further chromosome changes during tumor formation, suggesting activation of genes required for tumorigenicity but not cell transformation, which may be picked up in these studies. Normal diploid human cells of foreskin origin have been successfully transfected to determine whether single or multiple oncogene combinations are tumorigenic. We have shown that neither SV40 nor the activated human H-ras gene (EJ) are tumorigenic in human cells though the respective gene products are produced. Double and triple transfections are now in progress using drug selection, but as yet no morphologically transformed colonies have been found. (P)